It is thought that radiation causes a p53-dependent G1 phase delay in normal cells to allow additional time for repair of DNA damage. However, it has been reported that human fibroblasts, unlike other cell types, do not delay after irradiation, but arrest permanently in G1 phase with a senescence-like phenotype. We have shown that: 1) the "permanent" arrest is, in fact, reversible in a substantial population of the irradiated culture if adhesion interactions are disrupted; 2) the arrest is dependent on the type of extracellular matrix (ECM); 3) the maintenance of the arrest depends on an extracellular factor(s); and 4) arrest results in the expression of smooth muscle alpha-actin, characteristic of the differentiated myofibroblast. We propose that candidate extracellular factors are reactive oxygen species (ROS) and TGF-beta1. We suggest that radiation causes an increase in ROS, which activates TGF-beta1. Cells might then arrest because of the direct action of TGF-beta1, or through the binding of RGD sequences in latency associated peptide (LAP) or latent TGF-beta1 binding protein (LTBP) to growth inhibitory integrins. Fibroblasts might differentiate to myofibroblasts because of TGF-beta1 induction of ED-A fibronectin, a spliced form of fibronectin that has been shown to cause differentiation of fibroblasts in response to TGF-beta1. The ECM dependency of arrest could result from a preferential activation, expression, or localization of these proteins. These studies are important because the effect of the extracellular environment on radiation arrest is rarely examined. Also, these studies are directly relevant to, and will have therapeutic impact on the treatment of radiation fibrosis because they focus on how the production and interplay of ROS and TGF-beta1 affect radiation cell cycle arrest and differentiation of fibroblasts in response to ECM signals. For example, TGF-beta1-induced myofibroblast differentiation is thought to play an important role in radiation fibrosis. In fact, clinical studies show that increased amounts of TGF-beta1 and the presence of myofibroblasts are an indicator of fibrotic progression. In the first year of the proposed work, we will study the interactions of ROS and TGF-beta1, and begin experiments with TGF-beta1, LAP, and LTBP-1 measurements and immunolocalization. In the second year, we will conclude work on TGF-beta1, LAP, and LTBP-1 measurements and immunolocalization, and perform experiments concerning ED-A fibronectin. In year three, we will measure amounts and kinase activities of CDKs and CKIs under conditions of radiation arrest, or ROS or TGF-beta1 inhibition. In the fourth year, we will identify growth inhibitory integrin receptors that interact with LTBP-1 or LAP after irradiation.